RSC Advances

PAPER

Quaternary struc

aDipartimento di Scienze e Tec

Farmaceutiche-STEBICEF, Universita degli

Edicio 17, 90128 Palermo, Italy. E-mai

giampaolo.barone@unipa.itbIstituto EuroMediterraneo di Scienza e Tec

90139 Palermo, ItalycDipartimento DiSVA, Universita Politecnica

Ancona, Italy. E-mail: m.g.ortore@univpm.i

† Electronic supplementary informa10.1039/c5ra05144d

Cite this: RSC Adv., 2015, 5, 49871

Received 23rd March 2015Accepted 27th May 2015

DOI: 10.1039/c5ra05144d

www.rsc.org/advances

This journal is © The Royal Society of C

tures of GroEL and naıve-Hsp60chaperonins in solution: a combined SAXS-MDstudy†

A. Spinello,ab M. G. Ortore,*c F. Spinozzi,c C. Ricci,c G. Barone,*ab

A. Marino Gammazzaab and A. Palumbo Piccionello*ab

The quaternary structures of bacterial GroEL and human naıve-Hsp60 chaperonins in physiological

conditions have been investigated by an innovative approach based on a combination of synchrotron

Small Angle X-ray Scattering (SAXS) in-solution experiments and molecular dynamics (MD) simulations.

Low-resolution SAXS experiments over large and highly symmetric oligomers are analyzed on the basis

of the high-resolution structure of the asymmetric protein monomers, provided by MD. The results

reveal remarkable differences between the solution and the crystallographic structure of GroEL and

between the solution structures of GroEL and of its human homologue Hsp60.

Introduction

The determination of protein quaternary structures is one of thefundamental challenges of structural biology. For example, drugdesign, which is aimed at nding small molecules complemen-tary in structure and charge to a specic protein or biomoleculartarget, clearly requires knowledge of the target architecture.Although X-ray crystallography provides the atomic coordinatesof a protein, hence its high resolution structure, the actualprotein conformation in solution can be slightly or noticeablydifferent from the one in the crystal phase.1 Small Angle X-rayScattering (SAXS) of proteins in solution can be able toevidence these differences, albeit this technique cannot providehigh resolution protein structure details.2 Moreover, the SAXStechnique can be successfully applied also to giant proteins,which are oen difficult to crystallize. On the other side,MolecularDynamics (MD) canprovide an atomic view of small ormedium-sized proteins in thermodynamic equilibrium and inthe presence of explicit solvent and ions, hence under conditionsmost closely resembling those in solution, and therefore in vivo.Hence, the high resolution structure of giant proteins subunitsobtained by means of MD can be used to analyze SAXS experi-mental curves over the entire protein complex, or oligomer.3

nologie Biologiche, Chimiche e

Studi di Palermo, Viale delle Scienze,

l: antonio.palumbopiccionello@unipa.it;

nologia-IEMEST, Via Emerico Amari 123,

delle Marche, Via Brecce Bianche, 60131

t

tion (ESI) available. See DOI:

hemistry 2015

On these grounds, we have decided to investigate thequaternary structure of two chaperones of the Heat ShockProteins (Hsp) family, namely the bacterial 60 kDa GroEL andits human homologue Hsp60 in its naıve form. Hsps playcrucial roles in biosynthesis, folding/unfolding, transport andassembly of other proteins.4 GroEL, one of the most deeply andwidely studied Hsp protein, assists client protein's folding byforming a tetradecameric structure with a barrel shape obtainedby two heptameric rings.5 The folding process is also assisted bythe 10 kDa co-chaperonin GroES and by ATP to ADP hydrolysis.5

For many years the active form of this supramolecular foldingmachine was assumed as an asymmetric bullet-shapedGroEL14:GroES7 complex, while very recently a symmetricfootball-shaped GroEL14:(GroES7)2 complex was evidenced to bethe real folding chamber.6 In this new model, the binding of thesubstrate protein (SP) precedes the one of ATP. Therefore, thebarrel-shaped tetradecamer in its T-state (ATP-unbound) is thebasis for the recognition of SPs.6 To the best of our knowledge,the tetradecameric structure of GroEL's T-state in solution,under physiological conditions, has not yet been reported. Onthe other hand, the human homologue Hsp60 similarly workswith its co-chaperone Hsp10 in a ATP-mediated process, evenif Hsp60 shows less affinity for Hsp10 than the bacterialhomologue, forming preferentially heptameric rings instead ofthe barrel-shaped double-ring.7 The crystal structure of humanHsp60, in complex with Hsp10, was only recently obtained,showing a symmetric football-shaped assembly also for themammalian form.8Hsp60 has recently received a new biologicaland medical interest, being considered a promising target forthe treatment or the diagnosis of many diseases like cancer,inammation and autoimmune diseases.9 Notably, thesepathological conditions seem to be related to a cytosolic accu-mulation of Hsp60, also in its naıve form.10 Naıve-Hsp60 is

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Fig. 1 SAXS experimental profiles of GroEL (open circles, c ¼ 3 g L�1)with tentative fitting curves obtained from the crystallographic struc-tures indicated in the legend (top), and from a combination (bottom) oftetradecamers and heptamers from the 4AAR PDB structurecomputed by the GENFIT software.14

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characterized by the presence of a 26 peptides MitochondrialImport Sequence (MIS) linked at the N-terminus, which iscleaved during translocation of the nascent peptide to theorganelles. The oligomeric states of naıve-Hsp60 were recentlyfully investigated, revealing the presence of an equilibriumbetween stable heptameric and tetradecameric forms, in a widerange of concentrations as evidenced by different techniquessuch as Size-exclusion Chromatography (SEC), Dynamic andStatic Light Scattering (DLS and SLS, respectively), FluorescenceCorrelation Spectroscopy (FCS), Gel electrophoresis andpreliminary SAXS experiments.11 In particular, Blue NativePolyacrylamide Gel Electrophoresis of Hsp60 and of GroELperformed at several protein concentrations (see Fig. 4 and 5 ofref. 11) unambiguously show that while GroEL maintains itstetradecameric structure, naıve-Hsp60 is resolved into twobands that, based on the measured molecular masses, areattributed to the heptamers and tetradecamers. Despite naıve-Hsp60 is emerging as perspective drug's target and a diag-nostic tool, its detailed quaternary structure in solution, underphysiological conditions, has not been still deeply investigated.In this study MD simulations and SAXS experimental data,analyzed by the QUAFIT method,12 interplay in order to obtainthe quaternary structure of the protein in solution. In partic-ular, MD simulations have provided structural and dynamicdetails of single GroEL and Hsp60 subunits – derived fromcrystallographic data or conveniently adapted from crystallog-raphy – at the equilibrium, while QUAFIT has been able toderive the structure of the protein assembly, determined by thebest arrangement of both the rigid domains that constitute thesubunit and the subunits that form the oligomer, according to aproper point group symmetry. The combination of MD simu-lations and the advanced SAXS data analysis provided thequaternary structure of both GroEL and Hsp60 in solution.

Results and discussionFitting of SAXS data with PDB structures

SAXS measurements of GroEL and naıve-Hsp60 in solution havebeen performed at the ID2 beamline of the European Synchro-tron Radiation Facility (ESRF), as extensively described inExperimental section.

Both protein samples have been measured at the sameconcentration in solution and present SAXS proles comparablewith those previously obtained. In details, albeit curves at low qvalues appear quite similar, their features at intermediate q arerather different.11 Because several crystallographic structures ofGroEL are deposited in the Protein Data Bank, we have tenta-tively tted our experimental data with the form factor calcu-lated from those structures, by applying the SASMOL approach13

included in the GENFIT soware package,14 as reported in Fig. 1,upper panel. Such gure shows that all crystallographic struc-tures fail to satisfactorily represent GroEL in solution. In detail,the well-evident rst minimum observed at q z 0.5 nm�1 couldnot be tted at all. However, GroEL SAXS experimental curvelooks similar to the one obtained by Arai et al.,15 suggesting thatsignicant differences between solution and crystallographicGroEL structures exist and conrming the accuracy of our

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experimental results. Since both tetradecameric and heptamericGroEL PDB structures are reported, we have attempted to t theexperimental curve by combining the presence of both oligo-mers in solution. However, no combination of these quaternarystructures has been able to satisfactorily t the SAXS data (Fig. 1,bottom panel, see also Fig. S5 and S6†).

In particular, while the position of the rst minimum in theq-range is well tted, its shape and intensity are not adequatelyreproduced. These preliminary tting approaches, which claimthat GroEL would not present in solution an equilibriumbetween heptamers and tetradecamers but a unique tetradeca-meric state,11 have prompted us to follow a different approachbased on the results of the MD simulations. In particular, wehave performed all-atom MD simulations on GroEL tetradeca-meric barrel-shaped structure (PDB ID: 4AAR), constituted by109 446 atoms and in the presence of 107 852 water moleculesin the simulation box, for 40 ns, in order to asses if structuralchanges, able to reproduce tting of SAXS data, could beobserved.

The relative root mean square deviations (RMSD) are shownin Fig. 2 for ring T (red line), for ring R (blue line), and for allatoms (black line), respectively. As expected, the biggest devia-tions are due to conformational changes of ring R (blue ring). Infact, seven ATP molecules are bound in this ring in the originalPDB structure and, as a consequence of the deletion of theseligands, the ring is subject to the conformational change R/ T.

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The other ring in the T state (red ring) is much more stable andthe RMSD slightly increases at the end of MD process, followingthe relaxation of the structure in a physiological environment.Evidently, a much longer simulation time is necessary to ach-ieve the equilibrium state of the whole protein in explicit watersolution.

Theoretical SAXS curves corresponding to several snapshotsalong the MD evolution of the crystallographic structure havebeen calculated by means of the SASMOL approach.13 However,for q < 2.0 nm�1 neither the SAXS curve of the structure at 12 ns,nor the one of the structure at 40 ns show remarkable differ-ences from the SAXS curve of the crystallographic 4AAR struc-ture. Of note, the largest differences between the theoreticalSAXS curves at 0, 12 and 40 ns occur at the high q-range, where itis known that the experimental curves are affected by noticeableerror bars. These remarks led us to conclude that the MDsimulation of the whole tetradecameric protein structure, beingextremely time consuming, is not a suitable procedure to reachan acceptable tting of SAXS data within reasonable time.

Fig. 3 RMSD plot of the Ca atoms of the GroEL subunit (top). Sche-matic representation of the relative domains motion along the firstthree eigenvectors (bottom). GroEL domains are highlighted bydifferent colors, i.e. equatorial (blue), intermediate (green) and apical(red) domains, respectively.

Structural analysis of GroEL

To provide the structure of the chaperonin GroEL in aqueoussolution under physiological conditions, we have exploited theknown exibility of the monomer of GroEL in the frame of itsquaternary structure16 and as isolated subunit.17 The basic ideawas to extract the monomer from the 4AAR PDB entry, investi-gate its exibility by means of MD simulations, and reconstructthe structure of tetradecamer by simultaneously nding thebest conformation of the MD modied exible monomer andthe best positioning of fourteen of such monomers, assembledaccording to the D7 point group symmetry, in order to get thebest t of the experimental SAXS curve (for a schematic repre-sentation of method's workow see Fig. S1†). To perform thistask we have exploited the QUAFIT soware package.12 It isworth to notice that the D7 symmetry is a combination of a C7

rotation axis with seven perpendicular C2 axes. According toprevious ndings,16 two exible linkers in the border regionbetween the apical and the intermediate domains can bechosen. In particular, the two linkers are composed by residues188–191 and 373–376, respectively. In order to corroborate this

Fig. 2 (a) RMSD plot of the non-hydrogen atoms, of ring T (red), ringtetradecameric structure. (c) SAXS theoretical curves obtained from sna

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initial hypothesis, we have performed 300 ns MD simulation onthe GroEL monomer extracted from the ring T of 4AAR PDBentry. The RMSD relative to the starting conguration is shownin Fig. 3, showing that the isolated subunit explores a wideconformational space, also in the absence of bound ATPmolecule. Monomer's exibility modes, evidenced by means ofthe principal component analysis (PCA),18 reveal the presence ofthree principal eigenvectors, out of y, relative to the mainconformational uctuations (see Fig. S2†). Representatively, therelative movements of these eigenvectors are schematicallydepicted in Fig. 3 (see the corresponding ESI Movies pca1.mpg,pca2.mpg and pca3.mpg† for a dynamic visualization).

Interestingly, in the isolated monomer the apical domain(shown in red in Fig. 3) can move with respect to the equatorial(blue) and intermediate (green) domains, which result to betightly bound, as already evidenced within the tetradecameric

R (blue) and the whole protein (black), respectively. (b) GroEL 4AARpshots along the MD simulation, as indicated in the legend.

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structure,16 thus conrming the choice of the exible linkers asthe hinge of these relative movements. Furthermore, a residueroot mean square uctuations (RMSF) analysis conrms thatselected sequences corresponded to uctuation minima at theinterface of the rigid domains (see Fig. S3†).

Using the QUAFIT method, according to the strategydescribed in the Experimental section, two different structuresof the tetradecamer (A and B), both able to best t the experi-mental SAXS curve, have been obtained.

Corresponding reduced c2 measuring the quality of datatting for A and B solutions are 1.38 and 1.13, respectively. Best

Fig. 4 SAXS experimental profiles of 3 g L�1 GroEL (open circles),compared with the curves obtained by QUAFIT for structure A (black)and B (blue). Top and side view, respectively, of tetradecamers andmonomers of A, B and 4AAR. GroEL domains are shown in red (apical),green (intermediate), blue (equatorial).

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tting curves and different views of the A and B structures arereported in Fig. 4.

By comparing tetradecamer's size, we could observe that,although the oligomers are quite similar concerning externaldimension (gyration's radius and barrel axes), major differ-ences are related to the size of the internal cavity which isconsiderably smaller for structures A and B than for 4AAR (seebelow).

This feature could be explained by comparing the monomersfrom each oligomeric structure. In fact, in solution the apicaldomain seems to adopt a tilted conformation, with respect tothat of the monomer extracted from 4AAR, orienting thisdomain toward the center of the barrel. Notably, these confor-mational changes are representative of the bending moderelative to the rst eigenvector evidenced by the PCA reported inFig. 3. Structural changes due to chain exibility, imposedduring the tting process, did not dramatically distort theprotein backbone, as evidenced by the Ramachandran's plot(Fig. 5). Representatively, non-covalent interactions in structureA were analyzed. Interestingly, the proposed solution structurepresents several new hydrogen bonds and salt bridges (seeFig. 6) between adjacent subunits of the same ring (intra-ringsalt bridges), involving residues Asn229–Glu238 in the apicalregion, and Lys80–Asp41 in the equatorial domain.

Notably, two repeated inter-ring hydrogen bonds were alsodetected between Asn437–Glu434, in the equatorial domain.Although is reported that the two rings communicate throughtwo interfacial sites (L and R) in the crystallographic struc-tures,16 in our structure we have found that the R site is lacking.Moreover the L site has different types of non-covalent bonds.The interfacial R site may be formed aerwards upon recogni-tion of the substrate protein. In Fig. 7 a section of the GroELcavity (PDB: 4AAR) is compared with that of the structure

Fig. 5 Ramachandran's plot for the crystallographic (4AAR, left) andthe solution structures A, B (right) of GroEL.

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Fig. 6 Inter-ring (left) and intra-ring (right) salt bridges between theGroEL subunits in solution; involved residues are represented as balls-and-sticks.

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obtained by SAXS experiments in solution, as described above.Two essential differences have been highlighted: (i) the size ofthe two access windows and (ii) the shape of the internalchamber, where the client unfolded protein is hosted forundergoing the folding process. In fact, the structure in solu-tion presents a smaller window of about 2 nm compared to thatof the high-resolution solid state structures, such as 4AAR.

This feature induces us to suggest a role of GroEL, not onlyfor recognition of client proteins, but also for the initial dena-turation step, forcing the nascent polypeptide to pass throughthis tighter entrance. On the other hand, the cavity's size wasestimated by counting the number of water molecules insideeach heptameric ring (see e.g. Fig. S4†) for 4AAR (6591), GroEL-A(4136), GroEL-B (3968), Hsp60-A (5147) and Hsp60-B (4349).These results show in solution a shrinking of available volumefor the client proteins.

Fig. 7 Cavities of 4AAR in the solid state (left), and of the solutionstructures of GroEL (center) and Hsp60 (right).

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Structural analysis of naıve-Hsp60

Due to the unavailability of high-resolution structure of thehuman Hsp60 monomeric subunit, the latter was reconstructedby homology modeling (see Experimental). Moreover, themissing residues (MIS and unresolved C-terminal aa) weremodeled through the ROSETTA soware (see Experimental).Following the same approach used to study the structure ofGroEL in solution, we have performed 200 ns of MD simulationon the reconstructed Hsp60 monomer. A representative snap-shot, at about 150 ns, is shown on the right of Fig. 8, with theattached MIS highlighted in cyan. The presence of these addi-tional C- and N-terminal sequences clearly impose an intrinsicstiffness to the monomer structure, as observed from the RMSDplot aer 120 ns (Fig. 8 le).

Similarly to the approach followed for GroEL reconstructionand considering the presence of the two added N- andC-termini, the Hsp60 monomer used within QUAFIT has beendivided in three rigid domains: the apical (shown in red inFig. 8), the combination of equatorial and intermediate frag-ments (blue and green) and the set of N- and C-endings foldedwith ROSETTA (cyan and gray).

Hence four exible linkers have been dened. Two of themare in the region between the apical and the intermediatedomains (as in the case of GroEL) and are composed by residuesfrom 211 to 214 and from 397 to 400, respectively. The other twoexible linkers, connecting the N- and C-ending domain to theequatorial–intermediate domains, are from residues 23 to26 and from 548 to 552. Taking advantage of recent ndingsabove mentioned,11 and considering initial attempt to ttingSAXS data (see Fig. S5†), we have considered that, unlike GroEL,naıve-Hsp60 in solution can simultaneously be present as amixture of tetradecamers and heptamers.

Consequently, the QUAFIT soware package allowed us toobtain best tting of experimental SAXS data with tetradeca-meric and heptameric structures using the exible monomermodel as their building block (Fig. 9). Nevertheless, we hadperformed QUAFIT analysis even in the hypothesis of a uniquetetradecameric population, but the bad tting quality (c2 > 2.3)conrmed the simultaneous presence of two quaternary struc-tures in solution. Two best structures (A and B) obtained with

Fig. 8 (a) RMSD plot of the Ca atoms of the naıve-Hsp60 subunit; (b)representative snapshot with the Hsp60 domains shown in red (apical),green (intermediate), blue (equatorial), the MIS and C-terminalsequences highlighted in cyan and in grey, respectively. The fourflexible linkers are also shown in black.

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QUAFIT are reported in Fig. 9, together with their correspond-ing best t curves (c2 are 1.28 and 1.31, respectively). Theequilibrium composition, expressed as the ratio between themolar concentrations of tetradecamer and heptamer, results tobe 0.58 � 0.02 and 0.70 � 0.05 for the A and the B solution,respectively.

Fig. 9 (a) SAXS experimental profiles of 3 g L�1 naıve-Hsp60 (opencircles), compared with the curves obtained by QUAFIT for structure A(black) and B (blue). Top and side view, respectively, of tetradecamersand monomers of A, B and 4AAR. Hsp60 domains are shown in red(apical), green (intermediate), blue (equatorial), MIS and C-terminalsequence are shown in cyan.

Fig. 10 Inter-ring (left) and intra-ring (right) salt bridges between theHsp60 subunits; involved residues are represented as balls-and-sticks.

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Due to the presence of the MIS sequences, the structures ofthe monomer A and B, seen in Fig. 9, result more compact thanthe ones for GroEL, conrming the stable RMSD values shownin the last 80 ns of the MD simulation for the isolated subunit.Also, for Hsp60 the entrance of the folding chamber is notablysmaller. Moreover, the MIS sequences are located inside thefolding cavity, thus leading apparently to a reduced spaceavailable for the guest proteins (see Fig. 7 and 9). Nevertheless,the presence of amino acidic chains inside the cavitiessurprisingly allows the entrance of a larger number of watermolecules compared to GroEL A and B (see above).

It has been reported that both cytosolic and mitochondrialHsp60 show chaperone activity in vitro,10 but this issue has notbeen fully addressed in terms of recognition ability of naıve-Hsp60. Nevertheless, the conformational changes due to thebinding of ATP, together with the capture and unfolding of thesubstrate protein on the apical domain, should dramaticallyadapt the cavity for the subsequent encapsulation of the guestprotein.

Non covalent interactions in structure A have been repre-sentatively analyzed. In particular, several salt bridges werefound (see Fig. 10) involving residues Glu281–Arg268 andAsp279–Lys269 (intra-ring salt bridges), in the apical region.Inter-ring salt bridges are also present in the equatorial domainbetween the following residues: Lys493–Asp452, repeated twice,and Glu129–Lys481.

Moreover, these data allow to better rationalize the model tobe used for computational studies in the frame of computer-aided drug-design on Hsp60 inhibitors. In fact, while for themt-Hsp60 the monomer represents the target, our ndingssuggest that for targeting Hsp60 in its naıve form an oligomericmodel should be used.

MethodsSAXS measurements

SAXS measurements were performed at ID2 beamline at theEuropean Synchrotron Radiation Facility (ESRF) in Grenoble,France. The explored q-range (q ¼ 4p sin q/l where 2q is thescattering angle and l ¼ 9.95 � 10�2 nm the X-ray wavelength)

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covers between 0.1 and 3 nm�1, being the sample to detectordistance set to 1.5 m. Experiments were carried out at 20 �C and37 �C using a sealed 2 mm diameter quartz capillary enclosedwithin a thermostatic compartment connected to an externalcirculation bath and a thermal probe for temperature control.Protein samples were prepared as previously reported.11 BothHsp60 and GroEL solutions were measured at the weightconcentrations c ¼ 3 g L�1. SAXS images have been collectedusing a 2D detector (Pilatus3 1M). Every measurement wasperformed for 100 ms, and followed by a dead time of 3 s inorder to avoid radiation damage. The same sample wasmeasured 60 times at each temperature in order to improve thesignal-to-noise ratio. Incident and transmitted intensities wererecorded to the purpose of obtaining data in an absolute scale.Normalized SAXS patterns were azimuthally averaged to get theone-dimension proles of scattered intensities. The proteinmacroscopic differential scattering cross section, dS/dU(q), wasdetermined by subtracting from the protein in solution signalthe one of the buffer, corrected by its volume fraction in theprotein solution. Final SAXS dS/dU(q) curves, obtained inabsolute scale, clearly evidenced that both GroEL and Hsp60show no difference in the temperature range 20–37 �C.

QUAFIT approach

QUAFIT is a computer code designed for determining theoptimum conguration of a macromolecular assembly of aprotein in solution by the analysis of SAXS or Small AngleNeutron Scattering (SANS) experiments.12 Starting from a pointgroup symmetry, the structure of the protein assembly isdetermined through a sequence of aggregative intermediatespecies. The calculation progresses evaluating the relativepositions and orientations of the asymmetric units constitutingthe monomer, considered as rigid domains connected by ex-ible linkers of a known sequence, best tting small-angle scat-tering data. The program controls and limits the overlap amongrigid domains, exible linkers and monomers and takes intoaccount the possible presence of oligomerization intermediatesin solution.

For GroEL reconstruction, according the MD results and onthe basis of the PDB 4AAR structure, two units constituting themonomer have been considered as rigid domains (RDs). Therst RD includes the N- and the C-terminus referred to asequatorial and intermediate domains, represented in green andblue in Fig. 3 and 4. It is dened from residue 1 to 187 and from377 and 524. The second RD includes the amino acids from 192to 372. It is the red apical domain seen in Fig. 3 and 4. As aconsequence, the two RDs result to be connected by two exiblelinkers (FLs, shown in black in Fig. 3), both arranged by 4 aminoacids. The rst FL, dened by the sequence DVVE, connects theresidue 187 of the rst RD to the 192 of the second RD. Theother FL (AGGV) connects the residue 372 of the second RD tothe 377 of the rst RD. This monomer can form tetradecamersbased on the symmetry point-group D7.

The reconstruction of the Hsp60 structure has been per-formed by dividing the monomer unit, constituted by 573residues, in three RDs. The rst RD includes N- and C-termini

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and encompasses residues from 1 to 22 and from 553 to 573.It called MIS domain and shown in cyan in Fig. 8 and 9. Thesecond RD is from residue 27 to 210 and from 401 to 547 (blueand green domains, called equatorial and intermediatedomains in Fig. 8 and 9). The third RD, from 215 to 396, is theapical red domain seen in Fig. 8 and 9.

Accordingly, four FLs are dened. The rst FL, sequenced asTRAY, connects the residue 22 of the rst RD to the 27 of thesecond one. The second FL (LEII) is from residue 210 of thesecond RD to the 215 of the third one. The third and the fourthFLs (LSDG and EIPKE, respectively) connect residues 396 (thirdRD) and 401 (second RD) and residues 547 (second RD) and 553(rst RD). These FLs are shown in black in Fig. 8. A uniqueoptimized structure of the monomer is assembled according tothe symmetry groups D7 and C7 to dene the quaternarystructure of the tetradecamer and the heptamer, respectively.

The set of geometrical parameters optimized by QUAFITincludes the three polar coordinates of the geometrical center ofeach RD, the three Euler angles dening the orientation of eachRD, the three dihedral angles that dene the conformation ofeach residue belonging to each FL (two Ramachandran anglesfor the backbone and one angle for the side chain group).Moreover, in the case of Hsp6, QUAFIT also optimizes themolecular fraction of monomers forming tetradecamer. Themaximum rank L of the spherical harmonics expansion of thepartial X-ray scattering amplitudes has been xed to 7. Contactdistances among pairs of RDs are expanded in series of Stone'srotational invariant up to a maxim rank L0 ¼ 3. SAXS curves havebeen analyzed in the whole range of q.

Molecular dynamics simulations

GroEL quaternary structure (PDB ID: 4AAR) was obtained fromthe protein data bank and ATP molecules were deleted. TheGroEL monomer used in the following simulations was takenfrom this structure. The tertiary structure of the Hsp60 mono-mer was predicted using Swiss-Model Soware.19 The missingmonomer residues were folded using ROSETTA modeling so-ware20 and then added to the N- and C-endings of the modelwith the soware maestro.21 The fragment library were obtainedfrom Robetta server.22

All MD simulations were carried out through the GROMACS4.6.5 soware package,23 by following a recently reportedprocedure.24 Amber ff99SB-ILDN force eld25 was used.

A triclinic box of TIP3P water molecules was added aroundthe protein to a depth of 0.7 nm on each side. The charge of theprotein was neutralized and other Na+ and Cl� ions were addedto set the solution ionic strength to about 0.20 M. Explicitsolvent simulations were performed in the isothermal–isobaricNPT ensemble, at a temperature of 300 K, under control of avelocity rescaling thermostat.26 The particle mesh Ewaldmethod was used to describe long-range electrostatic interac-tions.27 The time step for integration was 2 fs and all covalentbonds were constrained with the LINCS algorithm. There weretwo temperature coupling groups in these simulations, the rstfor the protein and the second for water and ions. Preliminaryenergy minimizations were run for 5000 steps with the steepest

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descend algorithm. During the equilibration, the proteinsystem was harmonically restrained with a force constant of1000 kJ mol�1 nm�2, gradually relaxed into ve consecutivesteps of 100 ps each, to 500, 200, 100 and 50 kJ mol�1 nm�2.RMSD were referred to the starting congurations of MD.

Principal component analysis (PCA) was obtained by diago-nalizing the covariance matrix, which is built from the atomicuctuations in a MD trajectory where overall translational androtational motions have been removed. The monomer back-bone atoms were used to construct the protein covariancematrices. Upon diagonalization of this matrix, a set of eigen-values and eigenvectors was obtained. The eigenvectors corre-spond to directions in a 3N-dimensional space, and motionsalong a single eigenvector correspond to concerted uctuationsof atoms. The eigenvalues of the covariance matrix representthe total mean square uctuation of the system along the cor-responding eigenvectors. If eigenvectors are ordered accordingto their decreasing eigenvalues, the rsts describe the largestscale correlated motions. The trajectory were analysed usingVMD soware.28

Conclusions

Hsps are readily emerging as therapeutic and diagnostic targetsand their comprehensive structural knowledge is an importanteld of structural biology, useful for the development of newdrugs and therapies. Despite the well-known bacterial GroELhas been extensively studied, many concerns remain about itsstructure and function in solution and, more importantly,under physiological conditions. On the other hand, little isknown about Hsp60 in its naıve form. Here we take advantage ofcombining SAXS and MD techniques for the resolution atatomic level of two oligomeric structure revealing some newstructural features of GroEL and naıve-Hsp60 in solution. Theproposed methodology, here applied to exible subunits, butalso suitable for rigid monomers, does not require largecomputational resources and is not excessively timeconsuming, particularly if compared to the all atom MD of acomplete tetradecameric structure.

Acknowledgements

We gratefully acknowledge nancial support from the ItalianMIUR within the “FIRB – Futuro in Ricerca 2012” Program –

Project RBFR12SIPT, and the CINECA award N. IsB07, year2013, under the ISCRA initiative, for the availability of highperformance computing resources and support. We thankPierluigi San Biagio and Silvia Vilasi for samples and for stim-ulating discussions.

Notes and references

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2 E. Occhipinti, P. L. Martelli, F. Spinozzi, F. Corsi,C. Formantici, L. Molteni, H. Amenitsch, P. Mariani,P. Tortora and R. Casadio, Biophys. J., 2003, 85, 1165;

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